The vitamin A metabolite retinoic acid (RA) regulates multiple biological functions by activating the ligand-inducible transcription factors termed retinoic acid receptors (RAR). Our previous studies surprisingly demonstrated that RA also serves as an endogenous ligand for another nuclear receptor, namely PPAR4, and thus that the hormone displays a dual transcriptional activity. In addition to nuclear receptors, hydrophobic compounds, such as RA, associate in cells with members of the family of intracellular lipid binding proteins (iLBP), some of which function to directly deliver specific compounds from the cytosol to particular nuclear receptors in the nucleus, thereby augmenting the transcriptional activities of their ligands. Our recent findings revealed that the partitioning of RA between its two receptors is regulated by two iLBPs, cellular retinoic acid binding protein II (CRABP-II) which delivers RA to RAR, and fatty acid binding protein 5 (FABP5), which shuttles this ligand to PPAR4. Here, we propose to further delineate the molecular mechanisms that underlie the dual transcriptional activity of RA, to investigate potential roles of the newly discovered cross-talk between retinoid and PPAR4 signalling in regulating lipid metabolism and energy homeostasis, and to explore the possibility that, by activating its two receptors, RA may be protective against the development of obesity and insulin resistance. Proposed experiments will address two Specific Aims. 1. Roles of the dual transcriptional activity of RA in adipose function. Recent reports implicated both RAR and PPAR4 in regulation of different aspects of adipocyte biology and lipid homeostasis. Proposed studies will utilize cultured adipocytes to delineate roles of the two RA receptors in adipocyte differentiation and in various aspects of adipose biology such as insulin responsiveness and lipid metabolism. In addition, a mouse model susceptible to obesity and diabetes will be employed to investigate the involvement of RA and its associated nuclear receptors and binding proteins in the development of obesity and insulin resistance in vivo. 2. Structural basis for the activation of PPAR4 by FABP5. Upon binding of RA, FABP5 is mobilized to the nucleus where it delivers the ligand to PPAR4. Interestingly, while FABP5 binds numerous lipophilic ligands, its nuclear translocation is triggered only by specific compounds. To gain insight into the structural features that underlie the ligand-specificity in activation of FABP5, we propose to solve and compare the crystal structures of the protein complexed with activating vs. non-activating ligands. In addition, to elucidate the structural basis for the ability of FABP5 to productively associate with PPAR4, we plan to solve the structure of the complex of FABP5 with the receptor. Finally, we will determine the structure of PPAR4 with its only currently known physiological ligand, RA. PUBLIC HEALTH RELEVANCE: NARRATIVE: The vitamin A metabolite retinoic acid regulates multiple biological functions by activating two nuclear receptors: the retinoic acid receptor (RAR), and the peroxisome proliferator-activated receptor 4 (PPAR4). We recently found that the partitioning of this hormone between its two receptors is regulated by two members of the family of intracellular lipid binding proteins (iLBP), cellular retinoic acid binding protein II (CRABP-II) which delivers RA to RAR, and fatty acid binding protein 5 (FABP5), which shuttles this ligand to PPAR4. This project aims to explore the roles of retinoic acid and its receptors and binding proteins in adipose tissue biology and in regulating energy homeostasis. A special focus would be placed on investigating the possibility that, by activating the two receptors, retinoic acid may possess protective activities against the development of obesity and insulin- resistance. In addition, we propose to delineate the structural features that underlie the ability of FABP5 and PPAR4 to cooperate in mediating the transcriptional activity of retinoic acid.